MX2009013417A

MX2009013417A - Covalent modification of proteins for the instantaneous visualisation thereof and subsequent characterisation through mass spectrometry.

Info

Publication number: MX2009013417A
Application number: MX2009013417A
Authority: MX
Inventors: Robert Winkler; Marco Arnulfo Mata Gomez; Matthew Thomas Yasui
Original assignee: Itesm
Priority date: 2009-12-09
Filing date: 2009-12-09
Publication date: 2011-06-15
Also published as: WO2011071359A2; WO2011071359A3

Abstract

The invention described discloses the covalent modification of proteins for the instantaneous visualisation and subsequent characterisation thereof. The chemical mechanism employed is based on nucleophilic addition. The mechanism of the chemical reaction is explained, together with the bonding of the latter to specific aminoacid residues. Identification and characterisation of stained proteins employing mass spectrometry may be carried out by virtue of changes in mass of the residues modified by Uniblue A. A significant advantage of the staining employed in this invention lies in it having shown high compatibility with mass spectrometry analysis, it being unnecessary in addition to remove the colourant from proteins following staining as in the case of Coomassie or silver staining. Moreover, the derivatisation of amino acids gives rise to a change in the proteolytic hydrolysis patterns which may increase peptide sequencing coverage. Furthermore, improvement in the sequencing coverage of MS/MS analysis may be achieved by taking into account the changes in mass of the terminal amino groups of the derivatised amino acids. Additionally, a shorter procedure for protein identification is presented. Using the methods proposed for staining and analysis of proteins, the time required for protein analysis may be substantially reduced whilst the quality of the analysis may be improved.

Description

COVALENT MODIFICATION OF PROTEINS FOR ITS INSTANT AND FURTHER DISPLAY CHARACTERIZATION THROUGH MASS SPECTROMETRY.

DESCRIPTION FIELD OF THE INVENTION The present invention relates broadly to the chemical modification of proteins. More specifically, it describes methods for chemically modifying a specific site in the peptide chains of the proteins.

OBJECT OF THE INVENTION The invention is a method for carrying out a covalent modification of proteins for an instantaneous visualization and its subsequent characterization using mass spectrometry.

BACKGROUND The first step of a protein analysis is generally the separation of the proteins in polyacrylamide gels with sodium dodecyl sulfate (SDS-PAGE) followed by the staining of the proteins. The bands corresponding to the protein of interest can be cut directly from the gel and analyzed by mass spectrometry. Coomassie blue staining is one of the most frequently used techniques for protein staining in SDS-PAGE gels, due to its high compatibility with mass spectrometry analysis. However, this technique requires several hours. In addition to that, the dye must be removed before preparing the proteins for mass spectrometric analysis, this procedure needs at least one hour [1]. In the literature, various stains have been described for the covalent modification of proteins [2], [3] but the subsequent application of these stains in the characterization of proteins by mass spectrometry is not described.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Schematic representation of the addition reaction of Uniblue A to an amino group of the protein.

Figure 2-a. Visualization of an SDS-PAGE gel stained with Uniblue A. Sample of bovine serum albumin (BSA).

Lane 1: Molecular marker.

Lane 2: Sample of ASB stained with Uniblue A Figure 2-b. Visualization of an SDS-PAGE gel stained with Coomassie blue.

Sample of ASB.

Lane 1: Molecular marker.

Lane 2: Sample of ASB stained with Coomassie blue.

Figure 3-a. Visualization of a SDS-PAGE gel stained with Uniblue A. Sample of Rituximab, a recombinant antibody.

Lane 1: Molecular marker.

Lane 2: Rituximab stained with Uniblue A.

Figure 3-b. Visualization of an SDS-PAGE gel stained with Coomassie blue.

Sample Rituximab, a recombinant antibody.

Lane 1: Molecular marker.

Lane 2: Rituximab stained with Coomassie blue.

Figure 4. Analysis of samples of Escherichia coli TOPIO cells with and without expression of a recombinant protein. This protein is a fusion protein, malE-lacZa, from the pMAL-c4x vector (New England Biolabs Inc., NEB). The theoretical molecular weight of this protein is 50871.3 Da, which corresponds to the blue line of the molecular marker.

Figure 4-a. Visualization of an SDS-PAGE gel stained with Uniblue A for the identification of the expression of a recombinant Escherichia coli protein.

Lane 1: Molecular marker.

Lane 2: Protein profile of recombinant Escherichia coli stained with Uniblue A. Lane 3: Protein profile of undyed recombinant Escherichia coli.

Figure 4-b. Visualization of an SDS-PAGE gel stained with Coomassie blue for the identification of the expression of a recombinant Escherichia coli protein. Lane 1: Molecular marker Lane 2: Protein profile of recombinant Escherichia coli stained with Uniblue A and Coomassie blue.

Lane 3: Protein profile of recombinant Escherichia coli stained with Coomassie blue.

Figure 4-c. Direct comparison between strains of Escherichia coli without and with expression of a fusion protein of 50871.3 Da. 1 minute rapid stain only with Uniblue A.

Lane 1: Molecular marker Lane 2: Protein profile of Escherichia coli TOP10 / pMAL-c4x that produces a MalE-lacza fusion protein, staining with Uniblue A Lane 3: Protein profile of Escherichia coli TOPIO without expression, staining with Uniblue A Figure 5-a. Mass spectrum. ASB peptides analyzed by MS / MS without modification.

Figure 5-b. Mass spectrum. ASB peptides analyzed by MS / MS with modification in lysine K (1).

Figure 5-c. Mass spectrum. Direct comparison between the peptides with Uniblue A, modification (above) and without modification (below). The ions of the N-terminal group (bions) are easier to detect due to the addition of the mass corresponding to Uniblue A (484.0 Da).

Figure 6. Schematic representation of the method of the invention and the preparatory stages of samples.

Figure 7. Schematic representation of fragments "a", "b" and "c" (N-terminal) and "x", "y" and "z" (C-terminal) of a peptide.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for chemically modifying a protein, particularly by the nucleophilic addition of a dye with at least one functional group, to react in a nucleophilic addition with the amino groups of the protein, preferably at the amino acids lysine of the peptide sequence; (for example a vinyl group), and optionally has at least one other ionizable functional group to give greater solubility to water staining, subsequently give greater solubility to the peptides and influences their ionization by mass spectrometry (for example a sulfate group); preferably the dye is Uniblue A (see figure 1). The chemical modification of specific sites in the sequence of a protein with the dye demonstrates certain advantages for its visualization in an SDS-PAGE gel.

The advantages are directly related to the visualization of the proteins in a short period of time, without noticeable modification of their apparent mass in the SDS-PAGE, to later allow their characterization by mass spectrometry, considerably reducing the time of analysis. Additionally, the modification of the peptides can help to obtain more significant results in the analysis of the peptides by changes in the properties of the peptide.

To carry out the proposed method, the following solutions are required: a) Buffer solution A. The pH range of this solution is 8-9.

Preferably it consists of a solution of sodium carbonate (100 mM) and sodium dodecyl sulfate (SDS) at 10% (w / v) in solution with bidistilled water. The SDS is an anionic detergent, which is capable of dissolving hydrophobic molecules. The function of the SDS is to denature the proteins causing a change in the structure (primary, secondary, tertiary or quaternary) giving rise to a linear structure. Another function of SDS is to negatively charge proteins. These two functions are important to carry out an adequate separation of the proteins in an SDS-PAGE gel. b) Coloring solution. It refers to a dye in solution, where the dye has a functional group to react in a nucleophilic addition with the amino groups of the protein, preferably at the lysine amino acids of the peptide sequence; and optionally has at least one other ionizable functional group such as sulfate, to give greater solubility to water staining, later to give greater solubility to the peptides and influences their ionization by mass spectrometry. Preferably, it is Uniblue A, at a concentration of 60 mM, dissolved in the buffer solution A. c) Reductive solution. This solution has a pH of 6.8, and consists of a solution of glycerol (20% v / v), Tris HC1 pH 6.8 (200 mM) and dithiothreitol (DTT) 20 mM. The reduction of a disulfide bond by DTT is carried out by two sequential reactions, in which a sulfhydryl group (- SH) present in the disulfide bond (covalent bond between two SH groups present in cistern residues) is exchanged. d) Alkylation solution. It consists of a solution of iodoacetamide (IAA) at a concentration of 550 mM in buffer A. The alkylation occurs by the covalent attachment between the iodoacetamide and the SH groups present in the cysteine residues. e) Positive control. 3 mg of lyophilized bovine serum albumin (BSA) are reconstituted in 1 mL of buffer solution A. One of the reasons why the ASB was chosen is because it is a very well characterized and studied protein, which allows it to be used as a model to carry out various experiments, in addition to its availability in the market at a low cost.

The protein samples to analyze must comply with the following characteristics. a) Be in solution with at least 50% buffer A. b) Have a protein concentration in at least 100 mg / mL of solution. c) Lack of amino compounds that can clog or react with the Uniblue A dye.

The protein samples to be characterized by the method proposed in this application have been classified as described below. a) Dry protein sample (lyophilized). b) Sample of proteins in solution in a sufficient concentration and a compatible buffer solution. c) Protein sample in solution in a low concentration and unsupported buffer, (say unsupported buffer solution as that containing amino groups, eg: Tris).

Depending on the origin of the sample containing the protein, a protocol (see figure 6) is presented for the preparation of the sample, in such a way that: - In samples of dried protein (1), the sample is dissolved (2) in a buffer solution A.

-In protein samples in a sufficient concentration and a compatible buffer solution (3), enter stage a) of the proposed method.

-In samples in solution in which there is a low concentration of proteins and others have a non-compatible buffer solution (5), optionally: -The proteins can be precipitated using TCA / acetone and subsequently resuspended in buffer solution A (6); this is carried out by mixing the TCA / acetone (lg TCA / mL acetone, previously cooled to 4 ° C) and the liquid protein sample in a 1: 9 ratio. The mixture is placed in a bath with ice at 4 ° C for 1-2 hours. Afterwards, the samples are centrifuged at 14,000 rpm at 4 ° C for 15 minutes. The supernatant is removed and the precipitate is washed with acetone at least three times, then it is centrifuged at 14,000 rpm for 5 min and the supernatant is removed. Once washed, the precipitate is resuspended in buffer A.

-The non-compatible solution can be eliminated by ultrafiltration and at the same time the proteins are concentrated and subsequently the proteins are recovered with the addition of buffer A.

The method for covalent modification of proteins for instant visualization and subsequent characterization using mass spectrometry comprises the following steps: a) Dye a sample containing the protein of interest in solution, with a dye solution with 1 functional group, to react in a nucleophilic addition with the amino groups of the protein, preferably in the amino acids lysine of the peptide sequence; and optionally has at least one other ionizable functional group to give greater solubility to water staining, later to give greater solubility to the peptides and influences their ionization by mass spectrometry, the coloring solution is Uniblue A dissolved in the buffer solution A in a concentration 60 mM. The resulting sample solution is heated at 100 ° C for 1 minute, avoiding to prolong the exposure of the sample at this temperature because there is a risk of degrading the protein. The chemical reaction also works at lower temperatures, but with a longer incubation time. b) Reduction of protein (8). This is done by adding the reducing solution to the sample solution obtained in Step a, in a ratio of 1: 1 volumes (sample solution: reducing solution) preferably and the subsequent incubation in a range comprised between 90 ° C to 100 ° C during a time of at least 1 minute; The solution obtained is called a reduced sample solution. c) Alkylation of the reduced sulfhydryl groups (- SH) (9) present in the cysteine residues. This step is carried out by adding the alkylation solution in a ratio of 10: 1 volumes (reduced sample solution: alkylation solution). The time for alkylation is preferably 5 minutes at room temperature. d) Separation of the proteins in a polyacrylamide-sodium dodecyl sulfate gel (SDS-PAGE) (10). The separation was carried out according to the standard protocol described by Sambrook 2001, applying an electric current of 150 V for 45 minutes. For this case, protein separation was carried out in 10 and 12.5% polyacrylamide gels (although the concentration of polyacrylamide used in the gels depends on the molecular weight of the proteins of interest to be separated). e) Cut the protein bands from the SDS-PAGE gel (11). This is done by cutting the region where the band corresponding to the protein of interest in the gel is located. Subsequently, the bands with the protein of interest are cut into smaller fragments (lmm x lmm). f) Remove the excess water present in the gel fragments in addition to fixing the proteins (12), this is carried out Fixing and dehydrating the gel fragments.

This is done by the direct addition of acetonitrile to the gel fragments until covered and incubated for 5 minutes at room temperature, then the excess acetonitrile is removed from the samples, and these are dried by vacuum centrifugation for 10 minutes. This stage is recommended for decrease the effects of acetonitrile on trypsin when digestion is performed.

Tryptic digestion of the proteins present in the fixed and dehydrated gel fragments (13). This is done by the addition of a trypsin solution (V51 1A Promega, 10 ng / μ ?, in NH4HCO3, 25 mM) sufficient to cover the previously fixed and dehydrated gel fragments. Subsequently, the gel fragments are incubated at a temperature of 60 ° C for 30 minutes, at this stage the hydrolysis reaction by trypsin near residues modified with Uniblue A is inhibited, therefore the hydrolysis reaction is mostly carried out in the arginine residues obtaining larger proteolytic fragments, achieving an improvement in the coverage of the peptide sequencing, it should be noted that in this stage the ions of the N-terminal group preceded by a Usina have an additional mass corresponding to Uniblue A (484.0 Da), which facilitates its detection by mass spectrometry, and each modification of Usina affects the mass of the amino acid fragments in the N or C terminal direction.

Extraction of the peptides from the gel fragments (14). This is carried out by the addition of a solution of acetonitrile / trifluoroacetic acid 0.1% (v / v) in a 1: 1 ratio to the gel fragments followed by incubation at 60 ° C for 15 minutes. In order to obtain a greater coverage of the peptide sequence, it is recommended to use extended extraction times. After extraction it is important to transfer the extraction solution to a new tube.

Dry the peptides (15), this is carried out by removing the excess acetonitrile / trifluoroacetic acid solution by vacuum centrifugation until dry peptides are obtained. j) Preparation and analysis of the peptides extracted by mass spectrometry (16). The extracted peptides are recovered by the addition of approximately 20 μl of a 0.1% (v / v) formic acid solution, the solution with the peptides is transferred into polypropylene vials (FAMOS) for nano LC-MS / MS. The analysis of the peptides can be performed, e.g. ex. in a nano LC-ESI-ion trap. The resulting spectrum can be evaluated using automatic investigation algorithms such as X! Tandem or OMSSA. To quantify the number of covalent modifications, the mass changes of + 484.03989 Da (monoisotopic) for Uniblue A. are taken into account.

Another object of the present invention relates to a kit for instantly visualizing proteins for further characterization using mass spectrometry. The trumpeted Kit includes the following reagents, stored independently: a) A buffer solution A. This solution has a pH in the range of 8 to 9, preferably consisting of a solution of sodium carbonate at a concentration of 100 mM and a solution of sodium dodecyl sulfate (SDS) at a concentration of 10% ( p / v). b) Uniblue A dye solution. The Uniblue A dye is dissolved in buffer solution A, at a concentration of 60 mM. c) Reductive solution. This solution has a pH of 6.8, and consists of a 20% (v / v) solution of glycerol, 200 mM Tris HC1 and 20 mM dithiothreitol (DTT). d) Alkylation solution: It consists of a solution of iodoacetamide (LAA) at a concentration of 550 mM in buffer A. e) Positive control: 3 mg of lyophilized bovine serum albumin (BSA), to be reconstituted in 1 mL of buffer solution A.

The instructions for using the Kit are described below: a) Add 10 iL of coloring solution to 90 μ? - of sample solution containing the protein of interest, to obtain a colored sample solution, b) Heat the colored sample solution at 100 ° C for 1 minute, c) Add 100 μl of the reducing solution to the hot colored sample solution, maintaining heating at 100 ° C for 1 minute, d) Add 20 iL of the alkylation solution to each hot colored sample solution of c), e) Incubate the colored sample solution of d) at room temperature for 5 minutes. f) Load the colored sample solution into the SDS-PAGE gel, g) Separate the proteins from the colored sample of f) by electrophoresis (for example 150 V for 45 minutes, Bio-Rad Protean3 Minigel 10% Acrylamide).

Each of the following examples refers to the visualization of the protein bands in an SDS-PAGE gel, comparing the Uniblue A and Coomassie blue stains, this to show the instantaneous visualization of the proteins for their subsequent characterization by means of spectrometry of MS / MS.

Example 1. Covalent modification of proteins for instantaneous visualization using the Kit subject of this invention in a sample of bovine serum albumin (BSA) and subsequent characterization by mass spectrometry.

I. Preparation of the sample.

Reconstitute 3.0 mg of lyophilized ASB in 1 mL of buffer solution A in an Eppendorf tube.

II. Method of use of the Kit.

Add 10 iL of coloring solution (Uniblue A) to 90 IL of the ASB solution (ratio of volumes 1: 9). Subsequently, the samples were incubated at 100 ° C for 1 minute. Then 100 μ ?, of the reducing solution (ratio of 1: 1 volumes) was added to the aforementioned mixture and incubated at 100 ° C for 1 minute. Finally, 20 μl of the alkylation solution was added and incubated for 5 minutes at room temperature.

III. Separation of the proteins in an SDS-PAGE gel.

For the separation of the samples, gels with a concentration of 12.5% acrylamide (0.75 mm thick) were used. Once the samples were ready, volumes of 2 μ ?. of the ASB sample in each of the gel lanes. Additionally, a Kaleidoscope ™ molecular marker was used from Bio-Rad (in this case a volume of 5 \ L was used). The separation of the proteins was carried out at a voltage of 150 V for 45 minutes (see FIGS. 2-a and 2-b).

IV. Preparation of the sample for characterization.

First the cutting of the bands corresponding to the proteins of interest of the SDS-PAGE gel was carried out. Subsequently, the bands with the protein of interest were cut into smaller fragments (approximately 1 mm x 1 mm). Then, acetonitrile was added to the gel fragments in order to fix and dehydrate the proteins present in the gel fragments. Finally the excess acetonitrile was removed from the samples, and subsequently these were dried by vacuum centrifugation for 10 minutes. Subsequently, the tryptic digestion of the proteins present in the gel fragments was carried out. This was done by the addition of a trypsin solution (V511A Promega, 10 ng / μg - in NH4HCO3, 25 mM), sufficient to cover the previously fixed and dehydrated gel fragments. Subsequently, the gel fragments were incubated at a temperature of 60 ° C for 30 minutes. After digestion, the next step was the extraction of the peptides from the gel fragments. This was accomplished by the addition of a solution of acetonitrile / trifluoroacetic acid 0.1% (v / v) in a 1: 1 ratio to the gel fragments followed by incubation at 60 ° C for 15 minutes.

After extraction, the solution was transferred to a new tube. Finally, the excess liquid was removed by vacuum centrifugation until dry peptides were obtained.

Characterization of the sample by mass spectrometry.

The extracted peptides were recovered by the addition of approximately 20 μ! _. of a 0.1% (v / v) formic acid solution, the solution with the peptides was transferred into polypropylene vials (FAMOS) for nano LC-MS / MS. The analysis of the peptides was carried out using a nano LC-ESI-ion trap. The resulting spectrum was evaluated by the use of automatic investigation algorithms X! Tandem and OMSSA. To quantify the number of covalent modifications, we took into account the change in mass of + 484.03989 Da (monoisotopic) in the amino acids modified by Uniblue A, (see figure 5-a, 5-b and 5-c).

Example 2. Covalent modification of proteins for instant visualization using the subject kit of this invention in a sample of a recombinant antibody (Rituximab).

I. Preparation of the sample.

For this procedure, an exchange of a non-compatible buffer solution was carried out with buffer A. First, 250 μ ?, of a recombinant Rituximab solution (500mg / 50mL) were placed in an Amicon® Ultra ultrafiltration tube (Millipore ™ ) with a capacity of 0.5 mL and a membrane of 3000 MWCO (Molecular Weight Cut Off, [Da]). Subsequently, 250 IL of buffer solution A was added.

The sample was then centrifuged at 14,000 x g for 30 minutes in a centrifuge for Eppendorf tubes. Then a sufficient volume of buffer A was added to achieve a volume of 0.5 mL (maximum capacity of the Eppendorf tube). Repeat this procedure at least five times. After repeating the previous procedure for the last time, Rituximab was resuspended in 125 μ ?, of buffer A, the final concentration of this solution was 20 mg / mL.

II. Method of use of the Kit. 10 μ? - of coloring solution (Uniblue A) was added to 90 μ ?, of the Rituximab solution (ratio of 1: 9 volumes). Subsequently, the samples were incubated at 100 ° C for 1 minute. Then 100 μl of the reducing solution (ratio of 1: 1 volumes) was added to the aforementioned mixture and incubated at 100 ° C for 1 minute. Finally, 20 xL of the alkylation solution were added and incubate for 5 minutes at room temperature.

III. Separation of the proteins in an SDS-PAGE gel.

For the separation of the samples, gels with a concentration of 12.5% acrylamide (0.75 mm thick) were used. Once the samples were ready, volumes of 2 μ ?, of the Rituximab sample were loaded in each of the lanes of the gel. A molecular marker Kaleidoscope ™ of BioRad was used (in this case a volume of 5 μ? - was used). The separation of the proteins was carried out at a voltage of 150 V for 45 minutes (see Figure 3-a and 3-b).

Example 3. Covalent modification of proteins for instantaneous visualization using it, in this case, in a sample of Escherichia coli, strain TOP10, with and without expression of a recombinant protein with the plasmid pMAL-c4x, which codes for a fusion protein .

The fusion is given by a maltose binding protein and the lacZa gene. In this example, an expression control of a protein was carried out.

I. Preparation of the sample.

The propagation of E. coli strains was carried out in 50 mL of TB Overnight Express medium (Novagen Inc.). Additionally, 150 μL of the antibiotic carbencillin was added to the TB medium for the recombinant strain to prevent the growth of other microorganisms. Then the culture medium was inoculated with 100 μL of an E. coli solution. The culture of E. coli was carried out for 16 hours at 37 ° C and an agitation of 250 rpm. After incubation, the culture medium with cells was separated in 7 mL volumes in 15 mL conical tubes and centrifuged at 5,000 x g at a temperature of 4 ° C for 15 minutes. Then the supernatant was removed and the precipitated E. coli cells, at the bottom of the tube, were resuspended with the addition of 4.5 mL of buffer A. Subsequently, the E. coli cell wall rupture was carried out for obtain the proteins present inside the cells, this was done by ultrasonication for 15 minutes. Afterwards, the sample was centrifuged at 10,000 x g for 15 minutes at 4 ° C, and the supernatant was recovered.

The proteins present in the supernatant were precipitated by the addition of 0.5 mL of a TC A / acetone solution (lg TCA / mL of acetone) in a 9: 1 volume ratio (Supernatant: TCA / acetone solution). This procedure was carried out at a temperature of 4 ° C for 1-2 hours. After precipitation, the samples were centrifuged at 10,000 x g for 15 minutes at 4 ° C. The supernatant was discarded and the precipitated proteins were washed with acetone (90%) at least three times (centrifuge at 10,000 x g for 15 minutes between each wash). Finally the excess acetone was removed and the proteins were reconstituted in 100 uL of buffer A.

Method of use of the Kit. 10 pL of the coloring solution (Uniblue A) was added to 90 μL · of the E. coli solution (ratio of 1: 9 volumes). The samples were incubated at 100 ° C for 1 minute. 100 μ ?, of the reducing solution (ratio of 1: 1 volumes) were added to the samples and then incubated at 100 ° C for 1 minute. Finally, 20 iL of the alkylation solution was added to the samples and incubated for 5 minutes at room temperature.

Separation of the proteins in an SDS-PAGE gel.

For the separation of the samples, gels with a concentration of 12.5% acrylamide (0.75 mm thick) were used. Once the samples were ready, they were loaded in volumes of 5 iL of the ASB sample in each of the lanes of the gel. A Kaleidoscope ™ molecular marker was used from BioRad (in this case a volume of 5 μ? Was used). The separation of the proteins was carried out at a voltage of 150 V for 45 minutes (4-a, 4-b and 4-c).

Table 1: Analysis of ASB peptide (2+ load) identified by OMSSA (tolerance 2 Da, 0.8 Da for fragments) ASB peptide sequence Sec ID No.1 K V P V V S T P T L V E V S R Lys Val Pro Glm Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg 1 5 10 15 Table 2: N-terminal fragments of the peptide without and with modification with Uniblue TO.

Sec. Code Frag. without modification with Uniblue A AA AA N-term Theoretical Theoretical measured Measured K (l) Lys bl 129.10 - 613.14 613.19 V Val b2 228.17 228.07 712.21 712.37 P Pro b3 325.22 325.15 809.26 - Q Glm b4 453.28 453.32 937.32 937.49 V Val b5 552.35 552.43 1036.39 1036.53 s Ser b6 639.38 639.52 1123.42 1123.59 T Thr b7 740.43 740.68 1224.47 1224.71 P Pro b8 837.48 - 1321.52 - T Thr b9 938.53 - 1422.57 - L Leu blO 1051.62 1051.88 1535.66 1535.66 V Val bl l 1 150.68 (1150.7) * 1634.72 1634.81 E Glu bl2 1279.73 1279.94 1763.77 1763.89 V Val bl3 1378.79 1378.89 1862.83 1862.93 s Ser bl4 1465.83 1465.73 1949.87 1950.63 ** R Arg bl5 - - - - * Identified only by OMSSA for the highest tolerance ** Manual analysis using mmass, error 0.76 Da Letter Normal style: manual analysis with mmass (tolerance 0.5 Da), Letter Bold style automatic analysis with OMSSA (tolerance 0.8 Da for fragments, optional modification with Uniblue A with 484.0 Da).

Table 3: C-terminal fragments of the peptide without and with modification with Uniblue A Sec. Code Frag. without modification with Uniblue A AA C- AA term Theoretical measured measured theoretical K (l) Lys yl5 - - - - V Val yl 151 151 1.84 1512.32 151 1.84 - P Pro yl3 1412.77 1412.98 1412.77 1413.04 Q Glm yl2 1315.72 1316.04 1315.72 1315.78 V Val yl l 1 187.66 1187.89 1 187.66 1187.94 s Ser ylO 1088.60 1088.79 1088.60 1088.79 T Thr y9 1001.56 1001.80 1001.56 1001.85 P Pro y8 900.52 900.74 900.52 900.71 T Thr y7 803.46 803.54 803.46 803.71 L Leu y6 702.42 702.42 702.42 702.35 V Val y5 589.33 589.55 589.33 589.53 E Glu y4 490.26 490.30 490.26 490.44 V Val y3 361.22 361.27 361.22 361.23 s Ser y2 262.15 262.06 262.15 - Arg and '175.12 - 175.12 -These results indicate that a molecular weight modification of 484.0 Da is present in position 1 of the peptide, Usina (lys, K).

Claims

CLAIMS Having sufficiently described my invention, I consider as a novelty and therefore, claim as my exclusive property, the content in the following clauses:

1. A method for covalent modification of proteins for their instantaneous visualization and subsequent characterization using mass spectrometry, characterized in that it comprises the steps of: a) Dye a sample containing the protein of interest in solution, with a dye solution with a functional group, to react in a nucleophilic addition with the amino groups of the protein, preferably in the amino acids Usina of the peptide sequence; and optionally has at least one other ionizable functional group to give greater solubility to water staining, subsequently to give greater solubility to the peptides and influences their ionization by mass spectrometry; b) Reduce the disulfide bonds of the protein contained in the sample solution obtained in step a), particularly by exchanging a sulfhydryl group (-SH) present in the disulfide bond by dithiothreitol; carrying this out by adding a reducing solution, preferably in a ratio of 1: 1 volumes (sample solution: reducing solution), and heating by incubation at 100 ° C for a time of at least 1 minute, the obtained solution is called reduced sample solution; c) Renting the sulfhydryl (- SH) groups of the reduced sample solution, present in the cysteine residues; this is carried out by adding an alkylation solution in a ratio of preferably 10: 1 volumes (reduced sample solution: alkylation solution) and maintaining at room temperature for at least 5 minutes; although the incubation time can be prolonged up to 5 minutes without affecting the reaction; the solution obtained is called the alkylated protein solution; d) Separate the proteins contained in the alkylated protein solution, in a polyacrylamide-dodecyl sulfate sodium gel (SDS-PAGE), according to the standard protocol described by Sambrook, applying an electric current preferably of 150 V for 45 minutes; it is possible to decrease the voltage and increase the separation time, as well as increase the voltage and decrease the separation time; e) Cut the bands of interest of the SDS-PAGE gel, in fragments of lmm x Imm; f) Remove excess water present in the gel fragments in addition to fixing the proteins, this is carried out by the direct addition of acetonitrile to the gel fragments incubate for 5 minutes at room temperature, then the excess acetonitrile is removed of the samples, and subsequently these are dried by vacuum centrifugation for 10 minutes; g) Tryptile digestion of the proteins present in the fixed and dehydrated gel fragments, this is done by covering the gel fragments, with a trypsin solution (V511A Promega, 10 ng ^ L in NH4HCO3, 25 mM), and incubate a temperature of 60 ° C for 30 minutes; h) Extract the peptides from the gel fragments digested in g), this is carried out by adding a solution of acetonitrile / trifluoroacetic acid 0.1% (v / v) in a 1: 1 ratio to the digested gel fragments, and incubate 60 ° C for 15 minutes; i) Dry the peptides, removing the excess acetonitrile / trifluoroacetic acid solution by vacuum centrifugation to obtain the dry peptides; j) Prepare the extracted peptides for analysis by mass spectrometry; the dry peptides are recovered by the addition of approximately 20 μ? - of a 0.1% (v / v) formic acid solution, the solution with the peptides is transferred into polypropylene vials (FAMOS) for nano LC-MS / MS .

The method for covalent modification of proteins according to claim 1, characterized in that in step a), the dye is preferably Uniblue A, at a concentration of 60 mM, dissolved in a solution of sodium carbonate (100 mM) and dodecyl sulfate sodium (SDS) at 10% (w / v) in solution with bidistilled water, at a pH in the range of 8 to 9.

The method for covalent modification of proteins according to claims 1 and 2, characterized in that in step a), the dyeing is preferably carried out by heating at 100 ° C for 1 minute; although the heating temperature may be less than 100 ° C, with long reaction times.

The method for covalent modification of proteins according to claim 1, characterized in that in step a), the sample containing the protein of interest, optionally consists of a: a) A sample with dry protein (lyophilized); b) A sample of proteins in solution in a sufficient concentration and a compatible buffer solution; c) A sample of protein in solution in a low concentration and unsupported buffer, (say unsupported buffer solution as that containing amino groups, eg: Tris).

The method for covalent modification of proteins according to claim 4, characterized in that the dried protein samples, the sample is dissolved in a solution of sodium carbonate (100 mM) and sodium dodecyl sulfate (SDS) at 10% (w / v) ) in solution with bidistilled water, at a pH in the range of 8 to 9.

The method for covalent modification of proteins according to claim 4, characterized in that the samples of proteins in solution in a sufficient concentration and a compatible buffer solution; they are used as is, in the proposed method.

The method for covalent modification of proteins according to claim 4, characterized in that the sample of protein in solution in a low concentration and non-compatible buffer solution; optionally they are: a) precipitated using TCA / acetone [lg TCA / mL acetone, previously cooled to 4 ° C] and subsequently resuspended in a buffer solution [the buffer solution consists of a sodium carbonate solution (100 mM) and sodium dodecylsulphate (SDS) at 10% (w / v) in solution with bidistilled water with a pH in the range between 8 to 9]. b) Ultrafiltered to eliminate the incompatible solution and at the same time concentrate the proteins and recover them with the addition of a buffer solution consisting of a solution of sodium carbonate (100 mM) and sodium dodecyl sulfate (SDS) at 10% (w / v) ) in solution with bidistilled water, at a pH in the range of 8 to 9.

The method for covalent modification of proteins according to claim 1, characterized in that in step a), in the dye solution the functional group for reacting in a nucleophilic addition is vinyl.

The method for covalent modification of proteins according to claim 1, characterized in that in step a), in the dye solution the ionizable functional group to give greater solubility to the water staining, subsequently give greater solubility to the peptides and influences their ionization by mass spectrometry to the peptides and solubility to staining in water, preferably it is sulfate.

10. The method for covalent modification of proteins according to claim 1, characterized in that in step g) the hydrolysis reaction by trypsin near residues modified with Uniblue A is inhibited, and because of this the hydrolysis reaction is carried out in most of the arginine residues obtaining larger proteolytic fragments, achieving an improvement in the coverage of peptide sequencing.

11. The method for covalent modification of proteins according to claim 1, characterized in that in step j), the ions of the N-terminal group preceded by a Usin have an additional mass corresponding to Uniblue A (484.0 Da), which facilitates its detection by mass spectrometry.

12. The method for covalent modification of proteins according to claim 1, characterized in that in step j), each modification of Usin affects the mass of the amino acid fragments in the N- or C-terminal direction.

13. The method for covalent modification of proteins according to claim 1, characterized in that in step j), the ions of the N-terminal group preceded by at least one Usin have a cumulative additional mass corresponding to Uniblue A (484.0 Da), to the plant.

14. The method for covalent modification of proteins according to claim 1, characterized in that in step j), the ions of the C-terminal group preceded by at least one Usin have a cumulative additional mass corresponding to Uniblue A (484.0 Da), to the plant.

15. A kit for instantly visualizing proteins for further characterization using mass spectrometry, characterized in that it comprises the following independently stored reagents: a) A buffer solution A, consisting of a solution of sodium carbonate at a concentration of 100 mM and a solution of sodium dodecyl sulphate (SDS) at a concentration of 10% (w / v), with a pH range of 8 to 9; b) Uniblue A dye solution, in which Uniblue A is dissolved in buffer A, at a concentration of 60 mM; c) Reductive solution, consisting of a 20% (v / v) solution of glycerol, 200 mM Tris HC1 and 20 mM dithiothreitol (DTT), with a pH of 6.8; d) Alkylation solution, consists of a solution of iodoacetamide (IAA) at a concentration of 550 mM in buffer A; e) Positive control: consists of 3 mg of lyophilized bovine serum albumin (BSA), with the instruction to reconstitute in 1 mL of buffer A.

16. A kit for instantly visualizing proteins for further characterization using mass spectrometry, according to claim 15, characterized in that the instructions are: a) Add 10 μ ?, of coloring solution to 90 μ ?, of sample solution containing the protein of interest, to obtain a colored sample solution, b) Heat the colored sample solution at 100 ° C for 1 minute, c) Add 100 μ ?, of the reducing solution, to the hot colored sample solution, maintaining the heating at 100 ° C for 1 minute, d) Add 20 μ ?, of the alkylation solution to each hot colored sample solution of c), e) Incubate the colored sample solution of d) at room temperature for 5 minutes, f) Load the colored sample solution into the SDS-PAGE gel, g) Separate the proteins from the colored sample of f) by electrophoresis (for example 150 V for 45 minutes, Bio-Rad Protean3 Minigel 10% Acrylamide).